Experimental Eye Research

Muller glia are retinal support cells that play crucial roles in tissue structure, waste management, and repair. A challenge for the field has been to find Muller glia markers that detect non-reactive cells or work well in non-mammalian models. We introduce two novel markers for identifying reactive and non-reactive Muller glia in vertebrate retinas: a modified enzyme lectin (GFP-EndoNDM) and a monoclonal antibody (mAb735). These markers recognize polysialic acid (polySia), which is a highly conserved glycosylation modification in humans and vertebrates. In the retina, polySia is present on Muller glia predominantly in the form of polySia-NCAM. We used GFP-EndoNDM and mAb735 to investigate polySia distribution in Muller glia of fish, amphibians, reptiles, birds, rodents, retinal organoids, and humans. In adult retinas of most species, polySia was localized to the Muller glia and spanned outer to inner retina, with inner plexiform layer (IPL) sublaminae ramifications. Gliosis was also detectable in degenerating murine retinas. Notable species differences were that only outer retinal regions of Muller glia were labelled in adult zebrafish, whereas the outer Muller glia body up to the first IPL sublamina was labelled in adult turquoise killifish. There was no significant retinal polySia labeling in larval zebrafish, but it was present in the brain. Larval turquoise killifish have polySia throughout the retina, similar to other adult vertebrates. Labelling polySia expands the scientific toolbox for Muller glia markers, and offers a versatile way to visualize and monitor structural changes in non-reactive and reactive Muller glia across most vertebrate species. HighlightsO_LIPolySia is a highly conserved Muller glia marker in vertebrate retina and retinal organoids C_LIO_LILabeling Muller glia with markers of polySia, facilitates the analysis of fine Muller glia processes in the IPL sublaminae, whole Muller glia morphology, and degenerative gliosis C_LIO_LIPolySia can be labeled with an engineered lectin conjugate (GFP-EndoNDM) and a commercially-available monoclonal antibody C_LI

PurposeGlaucoma, a multifactorial ocular neuropathy, can lead to irreversible vision loss. Diagnosis involves assessing optic cupping (increased cup-to-disc ratios) and structural changes (like retinal nerve fiber layer thinning) through clinical imaging. Elevated intraocular pressure (IOP) is commonly associated with glaucoma, but not always. However, understanding disease progression is hindered by limited access to donor ocular tissue and consistent clinical data. We hypothesize that the proteome of aqueous humor and plasma may be altered in disease and correlates with clinical parameters such as IOP and cup-to-disc ratios. MethodsAqueous humor (AH) and plasma samples were collected from 36 glaucoma patients (17 male, 19 female), and 35 non-glaucomatous control patients (16 male, 19 female) undergoing cataract surgery. The protein profile was compared using the SOMAscan(R) assay system for proteome profiling. From glaucomatous donors, correlations between IOP and cup- to-disc ratios to proteome differences were determined. ResultsOverall proteomics profiles between both AH and plasma were compared by combining all samples (glaucoma and non-glaucoma) and then performing correlation analyses. This study revealed similar protein abundance in the two biological fluids. Additionally, it identified different abundance of proteins in plasma and AH between glaucoma and non- glaucoma samples. The differential proteins identified were involved in pathways related to vascular integrity, inflammation, immune response, cell adhesion, and complement activation. Generally, glaucomatous AH showed higher protein levels. Neurofilament light chain (NEFL) protein correlated with elevated IOP and inflammatory markers, but not with cup-to-disc ratios. ConclusionsTogether, our data demonstrate that the proteins identified in this study from glaucomatous donors correspond to markers of neurodegeneration and those that may inhibit cell proliferation or disrupt vascular integrity.

PurposeLamina cribrosa (LC) deformations caused by elevated intraocular pressure (IOP) are believed to contribute to glaucomatous neuropathy and have therefore been extensively studied, in many conditions from in-vivo to ex-vivo. We compare acute IOP-induced global and local LC deformations immediately before (pre-mortem) and after (post-mortem) sacrifice by exsanguination. MethodsThe optic nerve heads of three healthy monkeys 12-15 years old were imaged with spectral-domain optical coherence tomography under controlled IOP pre-mortem and post-mortem. Volume scans were acquired at baseline IOP (8-10 mmHg) and at 15, 30, and 40 mmHg IOP. A digital volume correlation technique was used to determine the IOP-induced 3D LC deformations (strains) in regions visible pre-mortem and post-mortem. ResultsBoth conditions exhibited similar nonlinear relationships between IOP increases and LC deformations. Median effective and shear strains were, on average over all eyes and pressures, smaller post-mortem than pre-mortem, by 14% and 11%, respectively (Ps < 0.001). Locally, however, the differences in LC deformation between conditions were variable. Some regions were subjected pre-mortem to triple the strains observed post-mortem, and others suffered smaller deformations pre-mortem than post-mortem. ConclusionsIncreasing IOP acutely caused nonlinear LC deformations with an overall smaller effect post-mortem than pre-mortem. Locally, deformations pre-mortem and post-mortem were sometimes substantially different. We suggest that the differences may be due to weakened mechanical support from the unpressurized central retinal vessels post-mortem. Translational RelevanceAdditional to the important pre-mortem information, comparison with post-mortem provides a unique context essential to understand the translational relevance of all post-mortem biomechanics literature. PrecisThe authors compared in monkeys acute IOP-induced deformations of the lamina cribrosa pre-mortem and post-mortem. Deformation trends were similar pre-mortem and post-mortem, but deformations pre-mortem were generally smaller than those post-mortem, with substantial local variations. The differences are likely due to loss of vessel support post-mortem.

PurposeGrowing evidence suggests, in glaucoma, the dendritic degeneration of subpopulation of the retinal ganglion cells (RGCs) may precede RGCs soma death. Since different RGCs synapse in different IPL sublayers, visualization of the lamellar structure of the IPL could enable both clinical and fundamental advances in glaucoma understanding and management. In this pilot study, we investigated whether visible-light optical coherence tomography (vis-OCT) could detect the difference in the inner plexiform layer (IPL) sublayers thicknesses between small cohorts of healthy and glaucomatous subjects. MethodWe investigated vis-OCT retinal images from nine healthy and five glaucomatous subjects. Four of the healthy subjects were scanned three times each in two separate visits, and five healthy and five glaucoma subjects were scanned three times during a single visit. Raster speckle-reduction scans (3 by 3 by 1.2 mm^3: horizontal; vertical; axial directions with 8192x8x1024 samplings, respectively) of the superior macular were acquired. IPL sublayers were then manually segmented using averaged A-line profiles. ResultsThe mean ages of glaucoma and healthy subjects are 59.6 +/- 13.4 and 45.4 +/- 14.4 years (p =0.02, Wilcoxon rank-sum test), respectively. The visual field mean deviation (MD) are -26.4 to -7.7 dB in glaucoma patient and -1.6 to 1.1 dB in healthy subjects (p =0.002). The mean circumpapillary retinal nerve fiber layer (RNFL) thicknesses are 59.6 +/- 9.1 m in glaucoma and 99.2 +/- 16.2 m in healthy subjects (p=0.004). Median coefficients of variation (CVs) of intra-session repeatability for the entire IPL and three sublayers are 3.1%, 5.6%, 6.9%, and 5.6% in healthy subjects and 1.8%, 6.0%, 7.7%, and 6.2% in glaucoma patients, respectively. The mean entire IPL thicknesses are 36.2 +/- 1.5 m in glaucomatous and 40.1 +/- 1.7 micrometer in healthy eyes (p=0.003, Mixed-effects model). We found that the middle sublayer thickness was responsible for the majority of the difference (14.2 +/- 1.8 m in glaucomatous and 17.5 +/- 1.4 in healthy eyes, p<0.01). ConclusionsIPL sublayer analysis revealed that the middle sublayer could be responsible for the majority of IPL thinning in glaucoma. Vis-OCT quantified IPL sublayers with good repeatability in both glaucoma and healthy subjects. Visualization of the IPL sublayers may enable the investigation of lamella-specific changes in the IPL in glaucoma and may help elucidate the response of different types of RGCs to the disease.

The iris is a muscular organ whose deformations can cause primary angle-closure glaucoma (PACG), a leading cause of blindness. PACG risk assessment does not consider iridial biomechanical factors, despite their expected influence on iris deformations. Here we exploited an existing biometric data set consisting of near-infrared movies acquired during the pupillary light reflex (PLR) as a unique resource to study iris biomechanics. The PLR caused significant (>100%) and essentially spatially uniform radial strains in the iris in vivo, consistent with previous findings. Inverse finite element modeling showed that sphincter muscle tractions were c. 5-fold greater than iridial stroma stiffness (range 4- to 13-fold, depending on sphincter muscle size). This muscle traction is greater than has been previously estimated, which may be due to methodological differences and/or to different patient populations in our study (European descent) vs. previous studies (Asian); the latter possibility is of particular interest due to differential incidence rates of PACG in these populations. Our methodology is fast and inexpensive and may be a useful tool in understanding biomechanical factors contributing to PACG.

During corneal wound healing, stromal keratocytes transform into a repair phenotype that is driven by the release of cytokines, like transforming growth factor-beta 1 (TGF-{beta}1) and platelet-derived growth factor-BB (PDGF-BB). Previous work has shown that TGF-{beta}1 promotes the myofibroblast differentiation of corneal keratocytes in a manner that depends on PDGF signaling. In addition, changes in mechanical properties are known to regulate the TGF-{beta}1-mediated differentiation of cultured keratocytes. While PDGF signaling acts synergistically with TGF-{beta}1 during myofibroblast differentiation, how treatment with multiple growth factors affects stiffness-dependent differences in keratocyte behavior is unknown. Here, we treated primary corneal keratocytes with PDGF-BB and TGF-{beta}1 and cultured them on polyacrylamide (PA) substrata of different stiffnesses. In the presence of TGF-{beta}1 alone, the cells underwent stiffness-dependent myofibroblast differentiation. On stiff substrata, the cells developed robust stress fibers, exhibited high levels of -SMA staining, formed large focal adhesions (FAs), and exerted elevated contractile forces, whereas cells in a compliant microenvironment showed low levels of -SMA immunofluorescence, formed smaller focal adhesions, and exerted decreased contractile forces. When the cultured keratocytes were treated simultaneously with PDGF-BB however, increased levels of -SMA staining and stress fiber formation were observed on compliant substrata, even though the cells did not exhibit elevated contractility or focal adhesion size. Pharmacological inhibition of PDGF signaling disrupted the myofibroblast differentiation of cells cultured on substrata of all stiffnesses. These results indicate that treatment with PDGF-BB can decouple molecular markers of myofibroblast differentiation from the elevated contractile phenotype otherwise associated with these cells, suggesting that crosstalk in the mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB can regulate the stiffness-dependent differentiation of cultured keratocytes. Statement of SignificanceIn vitro experiments have shown that changes in ECM stiffness can regulate the differentiation of myofibroblasts. Typically, these assays involve the use of individual growth factors, but it is unclear how stiffness-dependent differences in cell behavior are affected by multiple cytokines. Here, we used primary corneal keratocytes to show that treatment with both TGF-{beta}1 and PDGF-BB disrupts the dependency of myofibroblast differentiation on substratum stiffness. In the presence of both growth factors, keratocytes on soft substrates exhibited elevated -SMA immunofluorescence without a corresponding increase in contractility or focal adhesion formation. This result suggests that molecular markers of myofibroblast differentiation can be dissociated from the elevated contractile behavior associated with the myofibroblast phenotype, suggesting potential crosstalk in mechanotransductive signaling pathways downstream of TGF-{beta}1 and PDGF-BB.

Postnatal ocular growth is regulated by a vision-dependent mechanism, termed emmetropization, which acts to minimize refractive error through coordinated growth of the ocular tissues. Many studies suggest that the ocular choroid participates in the emmetropization process via the production of scleral growth regulators that control ocular elongation and refractive development. To elucidate the role of the choroid in emmetropization, we used single-cell RNA sequencing (scRNA-seq) to characterize the cell populations in the chick choroid and compare gene expression changes in these cell populations during conditions in which the eye is undergoing emmetropization. UMAP clustering analysis identified 24 distinct cell clusters in all chick choroids. 7 clusters were identified as fibroblast subpopulations; 5 clusters represented different populations of endothelial cells; 4 clusters were CD45+ macrophages, T cells and B cells; 3 clusters were Schwann cell subpopulations; and 2 clusters were identified as melanocytes. Additionally, single populations of RBCs, plasma cells and neuronal cells were identified. Significant changes in gene expression between control and treated choroids were identified in 17 cell clusters, representing 95% of total choroidal cells. The majority of significant gene expression changes were relatively small (< 2 fold). The highest changes in gene expression were identified in a rare cell population (0.11% - 0.49% of total choroidal cells). This cell population expressed high levels of neuron-specific genes as well as several opsin genes suggestive of a rare neuronal cell population that is potentially light sensitive. Our results, for the first time, provide a comprehensive profile of the major choroidal cell types and their gene expression changes during the process of emmetropization as well as insights into the canonical pathways and upstream regulators that coordinate postnatal ocular growth.

Phagocytosis of retinal rod and cone outer segment (OS) tips by the retinal pigment epithelium (RPE) occurs daily to prevent accumulation of harmful compounds in the photoreceptors. Rhythmic bursts of phagocytosis, seen as increased levels of phagocytosed OS particles in the RPE, are known to appear once or twice a day depending on the animal species. However, differences in the rhythmicity of phagocytosis between distinct photoreceptor types are not well understood. Here, we show that phagocytosis of cone subtype OSs does not have identical rhythmic profiles in young zebrafish larvae. We investigated this by immunolabelling histological sections from the eyes of larvae that were collected at seven different time points throughout a 24 h circadian cycle. Internalized OS particles were then quantified from confocal images. The results revealed that OSs of all cone subtypes are phagocytosed continuously at some levels in young zebrafish. Interestingly, we observed a significant increase in the OS phagosome numbers from UV and blue cones at two time points, whereas green and red cones were phagocytosed more evenly throughout the day. We also investigated whether this rhythmicity is regulated by the external light by keeping the larvae in constant darkness before sample preparation. We found that complete darkness condition dampened the peaks in OS phagosome numbers from UV and blue cones indicating that the rhythmicity is primarily driven by the external light rather than the intrinsic circadian clocks in young larval zebrafish. Our findings provide new understanding on the rhythmicity of cone OS phagocytosis and its regulation.

PurposeAfter stromal injury to the cornea, the release of growth factors and pro-inflammatory cytokines promotes the activation of quiescent keratocytes into a migratory fibroblast and/or fibrotic myofibroblast phenotype. Persistence of the myofibroblast phenotype can lead to corneal fibrosis and scarring, which are leading causes of blindness worldwide. This study aims to establish comprehensive transcriptional profiles for cultured corneal keratocytes, fibroblasts, and myofibroblasts to gain insights into the mechanisms through which these phenotypic changes occur. MethodsPrimary rabbit corneal keratocytes were cultured in either defined serum-free media (SF), fetal bovine serum (FBS) containing media, or in the presence of TGF-{beta}1 to induce keratocyte, fibroblast, or myofibroblast phenotypes, respectively. Bulk RNA sequencing followed by bioinformatic analyses was performed to identify significant differentially expressed genes (DEGs) and enriched biological pathways for each phenotype. ResultsGenes commonly associated with keratocytes, fibroblasts, or myofibroblasts showed high relative expression in SF, FBS, or TGF-{beta}1 culture conditions, respectively. Differential expression and functional analyses revealed novel DEGs for each cell type, as well as enriched pathways indicative of differences in proliferation, apoptosis, extracellular matrix (ECM) synthesis, cell-ECM interactions, cytokine signaling, and cell mechanics. ConclusionsOverall, these data demonstrate distinct transcriptional differences among cultured corneal keratocytes, fibroblasts, and myofibroblasts. We have identified genes and signaling pathways that may play important roles in keratocyte differentiation, including many related to mechanotransduction and ECM biology. Our findings have revealed novel molecular markers for each cell type, as well as possible targets for modulating cell behavior and promoting physiological corneal wound healing.

PurposeTo investigate the contribution of the Bruchs membrane and sclera tissues to the overall structural integrity of the ocular wall. MethodsTwenty-three human globes were subjected to biomechanical testing. A piece of sclera measuring 5 x 5 mm was carefully removed at the nasal region, 2 mm away from the optic nerve head. The intraocular pressure was increased at approximately 1 mmHg/s until Bruchs membrane-uvea-retina-tissue layer (BMUR) ruptured. Next, strips of sclera and Bruchs membrane-choriocapillaris (BMC) complex were isolated from the superior fundus region. Uniaxial tension tests were performed at a strain rate of 0.01/s and sampling rate of 15 Hz. The tangent moduli of the BMC and sclera at 0.01, 0.02 and 0.03 strains were compared. ResultsThe rupture pressure of the BMUR was 98.1 {+/-} 21.4 mmHg. The tangent moduli of the BMC at 0.01, 0.02 and 0.03 strains were 2.96 {+/-} 1.44 MPa, 7.68 {+/-} 1.78 MPa and 9.43 {+/-} 2.11 MPa, respectively, and the tangent moduli of the sclera at 0.01, 0.02 and 0.03 strains were 1.09 {+/-} 0.80 MPa, 2.72 {+/-} 1.67 MPa and 5.69 {+/-} 3.27 MPa, respectively. ConclusionThe BMUR was able to sustain relatively high IOP before rupturing. The uniaxial tensile tests showed that the BMC tangent moduli were about 3 times of those of the sclera at strains of 0.01 and 0.02. Although the sclera is approximately 47 times thicker, the BMC is still likely to make a significant contribution (3.51% to 7.42% at strain <0.03) to the overall structural strength of the ocular wall.

PurposeWe aimed to assess optic nerve head (ONH) deformations following acute intraocular pressure (IOP) elevations and horizontal eye movements (adduction and abduction) in control eyes, highly myopic (HM) eyes, HM eyes with glaucoma (HMG), and eyes with pathologic myopia alone (PM) or PM with staphyloma (PM+S). MethodsWe studied 282 eyes, comprising of 99 controls, 51 HM, 35 HMG, 21 PM and 75 PM+S eyes. For each eye, we imaged the ONH using spectral-domain optical coherence tomography (OCT) under the following conditions: (1) primary gaze, (2) 20{degrees} adduction, (3) 20{degrees} abduction and (4) primary gaze with acute IOP elevation (to ~35 mmHg) achieved through ophthalmodynamometry. For each OCT volume, we automatically segmented the ONH tissues using deep learning. We performed digital volume correlation (DVC) analysis to compute IOP- and gaze-induced ONH displacements and effective strains (i.e. local deformations). All biomechanical quantities were compared across groups. ResultsUnder IOP elevation, we found that HM eyes exhibited significantly lower strains (3.9 {+/-} 2.4 %) than PM eyes (6.9 {+/-} 5.0%, p < 0.001), HMG eyes (4.7 {+/-} 1.8%, p = 0.04) and PM+S eyes (7.0 {+/-} 5.2%, p < 0.001). Under adduction, we found that HM eyes exhibited significantly lower strains (4.8% {+/-} 2.7%) than PM+S eyes (6.0 {+/-} 3.1%, p = 0.02). We also found significant associations between axial length (or refractive error) and strains - eyes with higher axial length and greater myopia were associated with higher strains. IOP-induced strains were also positively correlated with adduction-induced strains. ConclusionWe found that HMG eyes experienced significantly higher strains under IOP elevations as compared to HM eyes. Additionally, PM+S eyes experienced highest ONH strains as compared to other groups under all biomechanical loads. Our preliminary findings suggest the possibility of using a simple biomechanical test to tease out the susceptibility of HM eyes to further develop glaucoma and/or staphyloma.

Age-related macular degeneration is a leading cause of central vision loss in the elderly. Early hallmarks of the disease include basal laminar deposit and choriocapillaris degeneration. The location and composition of sialoglycoconjugates in healthy and diseased choroid and disease-related lesions have not been thoroughly examined. This study utilized lectins to examine sialoglycoconjugates in human tissue, specifically Sambucus nigra/Elderberry Bark Lectin (EBL) and Maackia amurensis lectin II (MAL-II), to examine -2,6 and -2,3 sialic acids, respectively. EBL and MAL-II both label the choroid and basal laminar deposit, with slightly different patterns. Whereas MAL-II predominantly labels the choriocapillaris endothelium, EBL also labels Bruchs membrane and extracellular domains surrounding the vasculature (intercapillary pillars). EBL labeling overlaps with the distribution of complement factor H to a greater extent than MAL-II. After treatment with neuraminidase to remove terminal sialic acids, a battery of lectins was applied to sections of choroids. Lectins that recognize {beta}-galactose, N-acetyllactosamine, galactose ({beta}-1,3) N-acetylgalactosamine, and - or {beta}-N-acetylgalactosamine showed increased reactivity, including increased labeling of glycans in basal laminar deposits. This study provides insight into the location and partial identities of sialoglycoconjugates in the human choroid, with possible implications for the pathogenesis of macular degeneration.

Eyesight plays essential roles for the survival of most animal species, and diseases leading to blindness or partial vision loss in humans have an enormous impact in life quality. The complex structure of the eye makes it ideal for the study of structure-function relationships, and to accomplish integration of histology with other disciplines, connecting basic anatomical sciences to clinical medicine. The RGB trichrome consists of the sequential staining with three dyes, fast green (as a general protein stain), picrosirius red and alcian blue (for two main components of the extracellular matrix, collagen and glycosaminoglycans respectively). Notably, this combination of primary colors matches the physiological color detection capacity of the human trichromatic eye. Application of RGB stain to human eye samples and to the eye of two widely used animal models, the mouse and the zebrafish (Danio rerio), gives rise to brilliant staining of eye connective tissues, with a range of colors from red to magenta depending on the extracellular matrix composition, and producing highly contrasted tissue interfaces, facilitating the observation of tissue structures, as well as histomorphometric analysis. Interestingly, staining of the retina resulted in differential staining of the retinal layers in the three species analyzed. These results support the effectiveness of RGB as a reliable general staining method, complementary to routine basic hematoxylin and eosin (H&E), applicable to the study of the eye in human pathology, basic preclinical research, as well as in histo(patho)logy teaching.

PurposePreviously we identified POU6F2 as a genetic link between central corneal thickness (CCT) and risk of open-angle glaucoma. The present study is designed to characterize the POU6F2-positive retinal ganglion cells (RGCs). MethodsThe Thy1-YFP-H mouse was used to identify the structure of POU6F2-positive RGCs in the retina. In the retina of the Thy1-YFP-H mouse approximately 3% of the RGCs were labeled with yellow fluorescent protein. These retinas were stained for POU6F2 to identify the morphology of the POU6F2 subtypes in 3D reconstructions of the labeled RGCs. Multiple retinal cell markers were also co-stained with POU6F2 to characterize the molecular signature of the POU6F2-positive RGCs. DBA/2J glaucoma models were used to test the role of POU6F2 in injury. ResultsIn the retina POU6F2 labels 32.9% of the RGCs in the DBA/2J retina (16.1% heavily and 16.8% lightly labeled). In 3D constructions of Thy1-YFP-H positive RGCs, the heavily labeled POU6F2-positive cells had dendrites in the inner plexiform layer that were bistratified and appeared to be ON-OFF directionally selective cells. The lightly labeled POU6F2 RGCs displayed 3 different dendritic distributions, with dendrites in the ON sublaminae only, OFF sublaminae only, or bistratified. The POU6F2-positive cells partially co-stained with Cdh6. The POU6F2-positive cells do not co-stain with CART and SATB2 (markers for ON-OFF directionally selective RGC), SMI32 (a marker for alpha RGCs), or ChAT and GAD67(markers for amacrine cells). The POU6F2-positive cells were sensitive to injury. In DBA/2J glaucoma model, at 8 months of age there was a 22% loss of RGCs (labeled with RBPMS) while there was 73% loss of the heavily labeled POU6F2 RGCs. ConclusionsPOU6F2 is a marker for a novel group of RGC subtypes that are ON-OFF directionally selective RGCs that are sensitive to glaucomatous injury.

The anatomical differences between the retinas of humans and most animal models pose a challenge for testing novel therapies. Non-human primate (NHP) retina is anatomically closest to the human retina with the presence of a high acuity region called the fovea. However, there is a lack of relevant NHP models for retinal degeneration that can be used for preclinical studies of vision restoration. To address this unmet need we aimed to generate inducible NHP models of photoreceptor degeneration. We generated three cynomolgus macaque models using distinct strategies. We used two genetically targeted strategies using optogenetics and Crispr-Cas9 to ablate specifically rods to mimic rod-cone dystrophy. Additionally, we created an acute model by physical separation of the photoreceptors and retinal pigment epithelium using a polymer patch. Retinal degeneration was evaluated in all three models by in-life exams such as fundus imaging, optical coherence tomography, adaptive optics and electroretinography. In the genetic models we observed punctuate areas of degeneration in the injected area marked by disorganization of outer segments, loss of rod photoreceptors and thinning of the outer nuclear layer. In the acute model, the degeneration was faster and involved both rods and cones. Among the three distinct NHP models, the Crispr-Cas9 based approach was the most advantageous model in view of recapitulating disease specific features and its ease of implementation. The acute model however resulted in the fastest degeneration making it the most relevant model for testing end-stage vision restoration therapies such as stem cell transplantation.

PurposeImpairment of the trabecular meshwork (TM) is the principal cause of increased outflow resistance in the glaucomatous eye. Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) are emerging as potential mediators of TM cell/tissue dysfunction. Furthermore, YAP/TAZ activity was recently found to be controlled by the mevalonate pathway in non-ocular cells. Clinically-used statins block the mevalonate cascade and were shown to improve TM cell pathobiology; yet, the link to YAP/TAZ signaling was not investigated. In this study, we hypothesized that YAP/TAZ inactivation with simvastatin attenuates glucocorticoid-induced human TM (HTM) cell dysfunction. MethodsPrimary HTM cells were seeded atop or encapsulated within bioengineered extracellular matrix (ECM) hydrogels. Dexamethasone was used to induce a pathologic phenotype in HTM cells in the absence or presence of simvastatin. Changes in YAP/TAZ activity, actin cytoskeletal organization, phospho-myosin light chain levels, hydrogel contraction/stiffness, and fibronectin deposition were assessed. ResultsSimvastatin potently blocked pathologic YAP/TAZ nuclear localization/activity, actin stress fiber formation, and myosin light chain phosphorylation in HTM cells. Importantly, simvastatin co-treatment significantly attenuated dexamethasone-induced ECM contraction/stiffening and extracellular fibronectin deposition. Sequential treatment was similarly effective but did not match clinically-used Rho kinase inhibition. ConclusionsYAP/TAZ inactivation with simvastatin attenuates HTM cell pathobiology in a tissue-mimetic ECM microenvironment. Our data may help explain the association of statin use with a reduced risk of developing glaucoma via indirect YAP/TAZ inhibition as a proposed regulatory mechanism.

Neurodegenerative changes predominate in early stages of diabetic retinopathy but effective therapies are lacking. Insulin treatment decreases neurodegeneration and intranasal insulin has been shown to reach the central nervous system in neurodegenerative diseases like dementia. We tested the hypothesis that intranasal insulin can decrease retinal neurodegeneration using the C57BL/KsJ-db/db transgenic diabetic (db/db) mouse model. Compared to the non-diabetic wildtype mice given intranasal saline, we observed decreased electroretinogram b-wave and oscillatory potential amplitudes in db/db mice treated with intranasal saline but not in the db/db mice treated with 2 units of intranasal insulin daily over 10 weeks. When compared to the non-diabetic intranasal saline control, we also observed decreased outer retinal thickness in the db/db mice given intranasal saline but this effect was attenuated in the db/db mice treated with intranasal insulin. GFAP immunoreactivity and caspase cell count were similarly elevated in the db/db mice treated with intranasal saline but not intranasal insulin. Mean blood glucose measurements increased 30 minutes after both intranasal saline and insulin treatment. Transcriptomic analysis revealed downregulation of inflammatory and apoptotic genes in the retina of db/db mice treated with intranasal insulin when compared to saline. In summary, treatment with intranasal insulin prevents the depression of b-waves and oscillatory potentials, decreases the attenuation of outer retinal thickness, reduces caspase cell count and GFAP immunostaining, and downregulates the transcription of inflammatory and apoptotic genes in the retina of db/db mice without exerting peripheral glucose lowering effects. Taken together, our results suggest that intranasal insulin can reduce neurodegeneration in diabetic retinopathy by improving retinal neuronal function, decreasing reactive gliosis and cell death, and modulating the expression of inflammatory and apoptotic genes.

PurposeElevated intraocular pressure (IOP) due to increased outflow resistance through the trabecular meshwork (TM) is a major risk factor for primary open-angle glaucoma. Outflow through the TM is segmental, consisting of high flow (HF) and low flow (LF) regions. Here, we investigate how ocular hypertension impacts segmental outflow using a dexamethasone (DEX) mouse model and compare TM stiffness between HF and LF regions. MethodsNanoparticles containing DEX or vehicle were injected twice weekly in 2-4-month-old C57BL/6J mice (n=14), and IOP was measured weekly. At week 4, mouse eyes were perfused in vivo with fluorescent nanospheres to assess flow patterns and the circumferential percentage of high, intermediate, and low flow regions in each eye. Sagittal sections were collected from HF and LF regions, and atomic force microscopy (AFM) was used to measure tissue stiffness. Immunofluorescent labeling was used to compare fibronectin and -SMA protein levels. ResultsDEX treatment significantly elevated IOP by an average of 33.3% and altered tracer distribution but not the percentage of HF and LF regions around the circumference. No significant differences in TM stiffness were detected between DEX-treated and control mice, or between HF and LF regions. Increased fibronectin in LF regions of DEX-treated eyes suggested subtle TM structural changes that were not detected by AFM. ConclusionsDexamethasone alters segmental flow distribution and may impact cell contractility rather than ECM stiffness to cause IOP elevation in young mice. These findings better characterize the nature of segmental outflow and TM mechanics in this model of steroid-induced glaucoma.

Neurodegeneration in glaucoma patients is clinically identified through longitudinal assessment of structure-function changes, including intraocular pressure, cup-to-disc ratios from fundus images, and optical coherence tomography imaging of the retinal nerve fiber layer. Use of human post-mortem ocular tissue for basic research is rising in the glaucoma field, yet there are challenges in assessing disease stage and severity, since tissue donations with informed consent are often unaccompanied by detailed pre-mortem clinical information. Further, the interpretation of disease severity based solely on anatomical and morphological assessments by histology can be affected by differences in death-to-preservation time and tissue processing. These are difficult confounders that cannot be easily controlled. As pathogenesis and molecular mechanisms can vary depending on the stage and severity of glaucoma, there is a need for the field to maximize use of donated tissue to better understand the molecular mechanisms of glaucoma and develop new therapeutic hypotheses. Further, there is a lack of consensus around the molecular RNA and protein markers that can be used to classify glaucoma severity. Here, we describe a multiparametric grading system that combines structural measurements of the retinal nerve fiber layer with linear regression and principal component analyses of molecular markers of retinal ganglion cells and glia (RBPMS, NEFL, IBA1 and GFAP) to stratify post-mortem glaucoma eyes by the severity of disease. Our findings show that a quantitative grading approach can stratify post-mortem glaucoma samples with minimal clinical histories into at least three severity groups and suggest that this type of approach may be useful for researchers aiming to maximize insights derived from eye bank donor tissue.

PurposeRetinal neurodegeneration is difficult to monitor due to insensitive disease endpoints. Mitochondrial dysfunction and oxidative stress are promising early biomarkers of retinal ganglion cell (RGC) degeneration. This study investigates dynamics of flavoprotein fluorescence (FPF), a non-invasive mitochondrial oxidative stress measure, and sensitivity to early neurodegeneration and neuroprotection in vitro and in vivo. MethodsFPF activity in response to neurodegeneration and neuroprotection were characterized in vitro in wild-type (WT) and SARM1 knockout (SARMKO) human embryonic stem cell-derived RGCs with and without Vacor treatment over 24 hours and confirmed with mitochondrial reactive oxygen species (ROS) measures. Further FPF evaluation was explored in vivo using the optic nerve crush (ONC) model in WT and SARMKO mice to compare early RGC stress detection within rodent retinas. ResultsIn vitro FPF intensities in WT RGCs increased within 8 hours of degeneration induction, preceding significant mitochondrial ROS production. Neuroprotective SARMKO RGCs maintained comparable FPF and ROS levels following insult. In vivo FPF changes were not observed in WT and SARMKO mice over 4 days following ONC, while only early retinal thickening was observed from OCT. Early FPF and OCT changes were not reflective of late RGC survival observed from ex vivo RGC soma and axon counts. ConclusionsThese findings highlight differences in FPF sensitivity to mitochondrial stress between simplified in vitro systems and complex in vivo rodent retinas. This study demonstrates the potential of FPF as an early neurodegeneration and neuroprotection endpoint in vitro while identifying limitations and areas of development for its translatability to preclinical in vivo assessment.